A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays
December 30th, 2017
The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).
The overall goal of this procedure is a rapid and efficient method to dissect pupal wings of Drosophila suitable for immunodetections or PCR assays. The procedure described here was developed by the demand to find a fast and efficient method to obtain pupal wing samples suitable to perform PCR assays or immunodetection protocols. Using a wet paintbrush, gently remove zero-hour APF pupa from culture vials and place them in new vials to complete the development period.
For example, if using a pupa that is 24 hours APF, it would left in the vial only 22 hours. At 22 hours, transfer the pupa to the microscope slide with the double-sided tape adhered to it. This transference could be done using a paintbrush to avoid damaging the pupa.
The pupa should be positioned forming a row with dorsal sides up and cephalic ends facing the same direction. After this, return the microscope slide to a constant temperature and allow the pupa to complete the appropriate developmental time. In this case, it would be 24 hours.
The microscope slide with the double-sided tape will be a transitory dissection platform to remove the pupal case. During the two hours on the microscope slide, the pupal case will dry, making it easily breakable with forceps. The first step in dissection is to remove the pupa from the carcass.
Then the pupa will be transferred to a different slide with double-sided tape adhered to it. The carcass will be left behind. This pupa will be stuck with the ventral side facing upwards, placed in the middle of the tape.
Before performing the wing cut, the pupa must be covered with a solution according to the procedure using immunohistochemistry. This solution will be affixative and the wing will be freed from the cuticle. Using forceps, gently remove the operculum from the pupal case.
Break the case with forceps, making a line along the side of the pupa to the caudal end of it. With the appropriate illumination, it is possible to visualize the internal shape of the pupa, making an evident space just above the hinge of the pupal wing. This space serves as a guide to perform the opening without damaging the pupa.
As the case is opened laterally, it will be possible to remove portions of it, picking them up by the open border and carrying them to the opposite side where the double-side tape will hold them. At the end of the dissection, the pupa will be almost naked. Only a piece of case will be covering the posterior tip.
With the aid of the forceps, gently push down the remainder of the case. This move will raise the interior end of the pupa, allowing both the head and thorax to stay in a preferential position to be stuck to the subsequent step. Take a new slide with double-sided tape and invert it on the pupa.
Then press slightly to make the pupa stick to the tape and gently slip the slide in order to remove the pupa from the rest of the cases. Invert the microscope slide. The pupa will be stuck on the dorsal side at the level of their head or thorax area.
For immunohistochemistry use, cover the entire naked pupa with 4%paraformaldehyde. After 10 minutes in affixative, with the aid of scissors, make a little incision at the hinge region of the wing or near it. Allow the affixative to reach the wing epithelium.
Then after five minutes, tear with forceps from the cuticle borders, expanding the hole and enabling the release of the wing epithelium from the cuticle sac. Once the wing tissue is released, leave it in the paraformaldehyde solution until completing 30 minutes of fixation. The method of dissection described in this video can be used to prepare high-quality samples of Drosophila pupal wings from different development stages for many types of techniques.
The protocol could be performed to dissect one pupa or several pupae at the same time. This helps to accelerate the procedure, making it easier to reach a large number of wings dissected. To remove the naked pupa from the opened case, it is important that the posterior tip will be small enough.
Although it is possible to remove a row of four or five naked pupa at the same time, it is not a relevant point, being able to remove those remaining attached to their case in successive steps using a single double-sided tape. This is confocal image of a 24-to 30-hour APF wing. The staining shows the expression of patched protein, the canonical receptor of hedgehog, the nuclei and actin filaments.
A similar dissection process could be used to obtain larger wing samples to isolate mRNA. This mRNA can be used to synthesize cDNA which can be used in PCR reactions. Here we show the products obtained to detect the expression of patched and actin.
The protocol offers a straightforward method for obtaining pupal wing samples that retain the familiar morphology of the wing. From these preparations, it is possible to obtain stacks of images that could be projected as 2D or 3D images to investigate distribution and/or the enrichment of proteins during the differentiation of the wing. A similar protocol can be used to collect a large pupal wing sample necessary to synthesize cDNA, a molecule essential in PCR reactions.
This procedure provides an alternative and complementary approach to the already-published methods for pupal wing dissection.
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Overview
This article describes a rapid and efficient method for dissecting pupal wings of Drosophila, which are essential for various techniques like immunodetection and PCR assays. The procedure aims to facilitate the study of transcript and protein localization during development.
Key Study Components
Area of Science
- Neuroscience
- Developmental Biology
- Genetics
Background
- Understanding transcript and protein localization is crucial in developmental studies.
- Drosophila serves as a model organism for genetic and developmental research.
- Efficient sample preparation is necessary for accurate experimental results.
- Immunodetection and PCR are common techniques used in biological research.
Purpose of Study
- To provide a straightforward procedure for dissecting pupal wings.
- To enhance the efficiency of obtaining samples for immunodetection and PCR assays.
- To support research in transcript and protein localization during Drosophila development.
Methods Used
- Use of a wet paintbrush to handle pupae gently.
- Transfer of pupae to microscope slides with double-sided tape.
- Timing the transfer based on the age of the pupa.
- Minimizing damage to the pupa during the dissection process.
Main Results
- The method allows for the collection of intact pupal wing samples.
- Samples obtained are suitable for various downstream applications.
- The procedure is quick and minimizes stress on the samples.
- Facilitates accurate localization studies in Drosophila.
Conclusions
- The described method is effective for dissecting pupal wings.
- It supports the study of developmental processes in Drosophila.
- The technique can be adapted for various experimental needs.
Frequently Asked Questions
Executive Industry Relevance
Efficient sample preparation of Drosophila pupal wings enables reliable detection of transcript and protein expression, supporting target validation and mechanistic de-risking in early discovery. This method provides a rapid, reproducible workflow for generating high-quality biological material suitable for downstream assays, reducing variability in phenotypic screening and assay development. By standardizing wing dissection, research teams can improve predictive confidence in pathway analysis and prioritize targets with stronger mechanistic rationale.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses through localized detection of proteins like patched, a canonical hedgehog receptor, in developing wing tissue.
- Operational Value: Supports biological de-risking by allowing consistent sampling across developmental stages for pathway clarification.
- Predictive Value: Facilitates assessment of target engagement and expression patterns critical for lead identification and portfolio triage.
Screening & Assay Development
- Scientific Value: Produces intact pupal wing samples suitable for immunodetection and PCR assays, enabling quantitative readouts of gene and protein expression.
- Operational Value: Standardizes sample preparation, improving reproducibility and scalability for high-throughput screening campaigns.
- Assay Readiness: Generates material compatible with downstream molecular analysis, supporting reliable compound evaluation in phenotypic screens.
Translational & Preclinical Research
- Translational Continuity: Provides a disease-relevant system for studying morphogenesis, with direct applicability to developmental pathway modeling.
- Mechanistic De-risking: Allows visualization of protein distribution and enrichment during differentiation, informing target confidence.
- Preclinical Alignment: Supports biomarker assessment through detectable expression of markers like actin and patched in wing epithelium.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target validation through assay development to preclinical evaluation, offering a reusable sample preparation technique for Drosophila-based studies.
- Discovery Biology: Supports hypothesis testing and pathway clarification by enabling detection of vein cell communication and cross-vein formation during pupal development.
- Screening: Delivers assay-ready samples with consistent morphology, enhancing reliability in immunodetection and PCR-based readouts.
- Analytics: Enables quantitative measurement of transcript and protein levels, aiding comparative analysis across genetic or experimental conditions.
- Translational Research: Connects early discovery to preclinical validation through conserved developmental mechanisms observable in wing morphogenesis.
- Enterprise Reuse: Establishes a standardized dissection protocol applicable across multiple projects studying Drosophila developmental genetics.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing mechanistic ambiguity in developmental pathways.
- Operational Value: Enhances reproducibility and scalability of sample preparation, supporting consistent assay performance.
- Strategic Value: Improves go/no-go decisions by providing reliable data on target expression and localization.
- Portfolio Impact: Enables risk-adjusted advancement decisions through higher-quality mechanistic data from reproducible samples.
Implementation Considerations
- Requires basic expertise in Drosophila handling and microscopy.
- Relies on standard tools: wet paintbrush, forceps, microscope slides with double-sided tape, and 4% paraformaldehyde.
- Necessitates timing precision based on hours after pupal formation (APF) to match developmental stages.
- Adaptable across wing developmental stages but optimized for 24–30 hour APF pupae.
- Limited by the need for careful manual dissection to avoid tissue damage, though proficiency improves with practice.
